|
| Status |
Public on Aug 17, 2017 |
| Title |
RNA NMP Single Cell CS44 |
| Sample type |
SRA |
| |
|
| Source name |
T+ / Sox2+ FACS sorted cells from caudal ends of WT embryos
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
developmental stage: TS13
|
| Treatment protocol |
In order to obtain single cell suspensions from embryo tissue for FACS sorting, 10ul of 5% BSA in PBS was added to the pooled samples and 100ul of ice-cold 0.05% trypsin was added. A homogenous suspension was achieved by slow pipetting and the reaction was stopped with the addition of 200ul of 5% BSA in PBS. Before cell sorting on a FACS Aria II (Becton Dickinson), the cell suspension was filtered through a 35µm mesh. the sorted cells were collected in Eppendorf tubes containing filtered 5% BSA in PBS at 4C. Cells were pelleted by centrifugation and subsequently resuspended in 10ul PBS. The density and viability (>90%) were checked on a Luna cell counter (Logos Biosystems). The suspension was adjusted to a cell density of 250 cells/µl, resuspension buffer added (4.5:1 ratio of cells:buffer) and approximately 5µl were loaded onto a 5-10µm C1 IFC (Fluidigm).
|
| Growth protocol |
ES cell lines were cultured according to standard conditions. Cells were maintained on gelatinized plates and mitotically inactive primary mouse embryo fibroblasts with standard ESC medium, refreshed daily, containing 15% FCS and 1000 U/ml leukemia inhibitory factor (LIF, Chemicon ESG1107). ES cells were used to generate embryos via the tetraploid complementation assay.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The IFC was processed according to the Fluidigm mRNA-Seq protocol. A 1:4000 dilution of ERCC spike-ins (Ambion) were added to the lysis buffer. Upon visual inspection, 55 capture sites were called to contain cells. The amplified cDNA was measured using the Qubit DNA HS assay and a selection of samples was verified on a Bioanalyzer DNA HS chip (Agilent).
The final libraries for those 55 capture sites were prepared with the Nextera XT (Illumina) kit according to the protocol, pooled and sequenced on a NextSeq (Illumina) sequencer using 2x75bp read lengths.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
embryos obtained from tatraploid complementation of embryonic stem cells
FPKM_SingleCell.txt
Fluidigm Single-Cell mRNA-Seq
|
| Data processing |
For single-cell RNA-Seq data, those libraries with very few reads (2 samples with less than 1x10^6 reads) were excluded from further processing. Reads were mapped to the mm10 genome as previously described for the bulk RNA-Seq analysis with TopHat2 (version 2.1.0) (Kim et al., 2013) using bowtie (Langmead et al., 2009), providing refSeq annotations in gtf format (UCSC) and the options --no-mixed --no-discordant -g1 --mate-inner-dist 200 --mate-std-dev 100, without initial trimming of reads. We then calculated FPKM values using Cuffquant and Cuffnorm from the Cufflinks (version 2.2.1) (Trapnell et al., 2012) package. Downstream analysis was carried out using the R Monocle package (version 2.4.0) (Qiu, et al., 2017; Trapnell, et al., 2014), by reading in the FPKM, sample and gene attributes tables from the Cufflinks output.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file includes FPKM values for every gene across all single-cell RNA-Seq samples
|
| |
|
| Submission date |
Jun 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Frederic Koch |
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Developmental Genetics
|
| Street address |
Ihnestraße 63-73
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE93524 |
Antagonistic activities of Sox2 and Brachyury control the fate choice of neuro-mesodermal progenitors |
|
| Relations |
| BioSample |
SAMN07205064 |
| SRA |
SRX2895001 |
